In all eukaryotes, numerous pathways and signaling networks depend upon modification by polymers of the protein ubiquitin (Ub) to regulate the levels, localization, interactions, or activities of thousands of proteins. Eight structurally distinct types of polyUb are known that differ by the Ub-Ub linkage(s) in the chain. Each form of the modification is thought to be associated with only a subset of functional outcomes, suggesting that downstream receptors can distinguish the different polyUb topologies through selective binding. Many types of ubiquitin binding domains (UBDs) have been identified, but few are known to be linkage-specific and, among those that are, the basis for their specificity has only recently begun to be understood. We have described how clustering two or more UBDs within a protein or protein complex can confer high-affinity, linkage-selective interactions with polyUb. This model, linkage-specific avidity, explains the specific binding of K63-linked polyUb by receptor proteins involved in the assembly of repair foci at DNA double-strand breaks, and it also helps to account for the K48-polyUb binding preference observed with the deubiquitinating enzyme ataxin-3. In this proposal, we set out to extend and apply our findings along two major fronts. First, the principles of linkage-specific avidity will be applied to engineer polyUb binding proteins with enhanced selectivity and affinity, and with new specificities for novel forms of polyUb. We will develop high-affinity, high-selectivity binding proteins that recognize K63, K48, and K48/K63-mixed-linkage forms of polyUb. Versions of these binding proteins will be made to specifically recognize types of free polyUb chains that have been implicated as critical signals in NfkB activation and virally-induced innate immunity. Strategies that underlie the design of these proteins also will be used to discover the specificities and functions of UBDs that thus far have defied solution. Second, a set of modular, designed UBD motifs with varying affinities and linkage specificities will be used to systematically probe the role of linkage specificity in polyUb receptor function. We will focus on polyUb receptor proteins that have three very different functions: human Rap80 is needed to assemble the BRCA1 repair complex to sites of DNA double-strand breaks; ataxin-3 is a deubiquitinating enzyme with a likely role in protein quality-control and that is the causative agent of the polyQ neurodegenerative disease spinocerebellar ataxia 3; and the yeast Rad23, a prototypical UBL-UBA protein that brings polyUb-protein conjugates to the 26S proteasome for degradation. These proteins have in common that their functions require polyUb binding, but the importance of affinity and Ub-Ub linkage specificity in these processes is not known. We will address this issue by testing the effects of systematic alterations of polyUb binding properties. These experiments will provide the first experimental tests of the commonly-held view that the linkage-selectivity of (poly)Ub receptors directs downstream functions.